1. Field of Invention
The present invention relates to the microbiological industry, more specifically to a bacterium of the family Enterobacteriaceae in which expression of gene(s) associated with the flagella formation and motility cascade is/are deregulated, and a method for producing an L-amino acid by fermentation of the above bacteria in which expression of the flagella formation and motility cascade gene(s) is enhanced.
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765) and alteration of regulatory regions such as promoter, leader sequence and/or attenuator or others known to the person skilled in the art (see, for example, US20060216796 and WO9615246A1). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to the feedback inhibition by the resulting L-amino acid (see, for example, WO9516042A1, EP0685555A1 or U.S. Pat. Nos. 4,346,170, 5,661,012 and 6,040,160).
Strains useful in production of L-threonine by fermentation are known, including strains with increased activities of enzymes involved in L-threonine biosynthesis (EP0219027A2 or U.S. Pat. Nos. 5,175,107; 5,661,012; 5,705,371; 5,939,307), strains resistant to chemicals such as L-threonine and its analogs (WO0114525A1, EP301572A2, U.S. Pat. No. 5,376,538), strains with target enzymes desensitized to the feedback inhibition by the produced L-amino acid or its by-products (U.S. Pat. Nos. 5,175,107 and 5,661,012), and strains with inactivated threonine degradation enzymes (U.S. Pat. Nos. 5,939,307 and 6,297,031).
It is described in the literature that motile bacteria such as Salmonella typhimurium, Escherichia coli, Bacillus subtilis, Yersinia enterocolitica, and the like can exhibit different lifestyles and can be in a motile single-cellular (or planktonic state) or can be sedentary cells that use adhesive fimbriae to cluster together and form biofilms on a surface (O'Toole G. A. et al., Annu. Rev. Microbiol., 2000: 54, 49-79). Escherichia coli (E. coli) cells, for example, are highly motile during the post-exponential growth phase (Amsler C. D. et al., J. Bacteriol., 1993: 175, 6238-6244), and become aggregated or adhere to a surface by the induction of adhesive curli fimbriae when entering into the stationary phase (Olsén A. et al., Nature, 1989: 338, 652-655).
It is known that in E. coli both flagella formation and motility, and curli-mediated adhesion cascades are under the control of regulatory feedforward cascades, each with a master regulator at the top, which acts as a massive environmental signal integrator (Pesavento C. et al., Gen. Dev., 2011: 22, 2434-2446). The curli fimbriae control cascade is a module within the general stress response, for which the σS factor (sigmaS, RpoS) acts as the master regulator (Hengge-Aronis R. 2000. The general stress response in E. coli. In Bacterial stress responses (eds. G. Storz and R. Hengge-Aronis), pp. 161-178. ASM Press, Washington, D.C.). The CsgD protein was shown to be an essential activator for the curli structural gene operon (csgBAC) (Gerstel U. et al., Mol. Microbiol., 2003: 49, 639-654).
For flagellar expression and motility cascade, the master regulator is the FlhDC complex expressed from flhDC operon defined as the flagella class 1 operon. The flagella master regulator FlhDC functions as a hetero-oligomeric complex of the FlhD and FlhC proteins (the FlhD4C2 complex), the crystallographic structure of which, originated from E. coli, was resolved (Wang S. et al., J. Mol. Biol., 2006, 355:798-808). The FlhDC complex regulates transcription from several flagellar and non-flagellar operons in bacteria, and, in particular, it activates the expression of class 2 operons, which are responsible for the inner part of the flagella and various factors (FliA, FlgM) (Pesavento C. et al., Gen. Dev., 2011: 22, 2434-2446). After the FlgM factor is secreted, FliA is released to activate class 3 operons which encode the outer subunits of additional flagella proteins required for flagellar function and chemotaxis, as well as a number of proteins of still unknown function (Aldrigde P. D. et al., Gen. Dev., 2006: 20, 2315-2326).
Another key regulator of cell motility and curli fimbriae expression is bis-(3′-5′)-cyclic-diguanosine monophosphate (c-di-GMP) degrading phosphodiesterase (PDE), for example, YhjH and YciR (the latter in addition to c-di-GMP PDE activity also functions in opposite direction as diguanilate cyclase). Overproduction of c-di-GMP PDE was demonstrated to interfere with motility of enteric bacteria through strong inactivation of expression of curli fimbriae and the biofilm matrix component cellulose (Römling U. et al., Mol. Microbiol., 2005: 57, 629-639; Jenal U. and Malone J., Annu. Rev. Genet., 2006: 40, 385-407). In a particular case, YhjH was shown to play a positive role in motility (Ko M. and Park C., J. Mol. Biol., 2000: 303, 371-382).
The FliZ protein, which is expressed from a gene immediately downstream from fliA, was demonstrated to be a highly potent inhibitor of curli fimbriae formation in E. coli by interfering with the activity of σS factor which controls genes involved in curli expression (Pesavento C. et al., Gen. Dev., 2011: 22, 2434-2446). More specifically, FliZ transiently gives motility (and therefore a foraging strategy) priority over the general stress response as long as flagellar gene expression keeps going.
A bacterium of the family Enterobacteriaceae, which has been modified to attenuate expression of the csgBAC or/and csgDEFG operons (RU2338782) or so as to not produce type I fimbrial adhesin protein (EP1838726A1), has been employed for producing an L-amino acid, specifically, L-threonine.
Furthermore, it is known from WO2002097089A1 that flagella-containing microorganisms, in which one or more genes relating to the constitution of flagella or movement of flagella is/are inactivated or deleted, can be used as hosts for producing useful substances, for example, amino acids (WO2002097089A1).
Until now, there have been no reports highlighting positive effect of enhanced expression of bacterial genes (flhDC, yhjH and flit) involved in the flagella formation and motility cascade on the productivity of L-amino acids, and more specifically L-threonine and L-phenylalanine.